Abstract:

The present invention relates to compositions containing an extract of
processed Panax genus plant and saponin compounds isolated therefrom for
treat the diseases caused by vascular injury in human or mammal, the
method for treating said diseases using thereby and the use thereof. The
extract of processed Panax genus plant and saponin compounds isolated
therefrom shows suppressive effect on endothelial dysfunction and damage
of blood-retinal on HUVEC and retinal vascular endothelial cell.
Therefore, it can be used as the therapeutics or functional health food
for treating and preventing the diseases caused by vascular injury.

Claims:

1. A use of an extract of processed Panax genus plants so as to make a
ratio of ginsenoside (Rg3+Rg5+Rk1) to (Rb1+Rb2+Rc+Rd) of over 1.0 as an
active ingredient for the preparation of the medicament employed for
treating and preventing the diseases caused by vascular injury.

2. A use of an extract of processed ginseng obtained by the steps;
treating Panax genus plants with heat at the temperature ranging from 70
to 150.degree. C. for the period ranging from 2 to 6 hrs, as an active
ingredient for the preparation of the medicament employed for treating
and preventing the diseases caused by vascular injury.

3. The use according to claim 1, wherein said extract is extracted with
the solvent selected from the group consisting of water, lower alcohols,
acetone, ethyl acetate, chloroform, dichloromethane, and the mixtures
thereof.

5. A use of saponin compounds at least one or their combination selected
from the group consisting of ginsenoside Rg3, Rg5 and Rk1, as an active
ingredient for the preparation of the medicament employed for treating
and preventing the diseases caused by vascular injury.

6. The use according to claim 1, wherein said diseases caused by vascular
injury is ischemic diseases or diabetic complications.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. A method for treating or preventing the diseases caused by vascular
injury in mammals, wherein the method comprises administering a
therapeutically effective amount of extract of processed ginseng or
saponin compounds isolated therefrom as set forth in claim 1 into the
mammal suffering with the diseases caused by vascular injury.

12. A pharmaceutical composition comprising an extract of processed
ginseng and saponin compounds isolated therefrom as set forth in claim 1
as an active ingredient in an effective amount to treat and prevent the
diseases caused by vascular injury.

13. A functional health food comprising an extract of processed ginseng or
saponin compounds isolated therefrom as set forth in claim 1 together
with a sitologically acceptable additive for the prevention and
improvement of the disease caused by vascular injury.

14. The use according to claim 2, wherein said extract is extracted with
the solvent selected from the group consisting of water, lower alcohols,
acetone, ethyl acetate, chloroform, dichloromethane, and the mixtures
thereof.

16. The use according to claim 2, wherein said diseases caused by vascular
injury is ischemic diseases or diabetic complications.

17. A method for treating or preventing the diseases caused by vascular
injury in mammals, wherein the method comprises administering a
therapeutically effective amount of extract of processed ginseng or
saponin compounds isolated therefrom as set forth in claim 2 into the
mammal suffering with the diseases caused by vascular injury.

18. A pharmaceutical composition comprising an extract of processed
ginseng and saponin compounds isolated therefrom as set forth in claim 2
as an active ingredient in an effective amount to treat and prevent the
diseases caused by vascular injury.

19. A functional health food comprising an extract of processed ginseng or
saponin compounds isolated therefrom as set forth in claim 2 together
with a sitologically acceptable additive for the prevention and
improvement of the disease caused by vascular injury.

20. The use according to claim 5, wherein said diseases caused by vascular
injury is ischemic diseases or diabetic complications.

21. A method for treating or preventing the diseases caused by vascular
injury in mammals, wherein the method comprises administering a
therapeutically effective amount of extract of processed ginseng or
saponin compounds isolated therefrom as set forth in claim 5 into the
mammal suffering with the diseases caused by vascular injury.

22. A pharmaceutical composition comprising an extract of processed
ginseng and saponin compounds isolated therefrom as set forth in claim 5
as an active ingredient in an effective amount to treat and prevent the
diseases caused by vascular injury.

23. A functional health food comprising an extract of processed ginseng or
saponin compounds isolated therefrom as set forth in claim 5 together
with a sitologically acceptable additive for the prevention and
improvement of the disease caused by vascular injury.

Description:

BACKGROUND OF THE INVENTION

[0001]1. Technical Field

[0002]The present invention relates to compositions containing an extract
of processed Panax genus plant and saponin compounds isolated therefrom
for treat the diseases caused by vascular damage in human or mammal, the
method for treating said diseases using thereby and the use thereof. More
particularly, the present invention relates to a use of an extract of
processed ginseng and saponin compounds isolated therefrom with enhanced
pharmacological effects due to heat-treatment of a Panax genus plants so
as to make a ratio of ginsenoside (Rg3+Rg5+Rk1) to (Rb1+Rb2+Rc+Rd) of
over 1.0.

[0003]2. Background Art

[0004]Blood vessels provide oxygen and adequate nutrients to the cells of
all organs as a pathway of blood delivery and plays important roles in
maintaining cell homeostasis by eliminating wastes matter from the system
as a main organ. Arterial wall consists of three layers, i.e., tunica
intima, tunica media and tunica adventitia. Tunica intima consists of
endothelial cell and elastic fiber covering therewith and tunica media
consists of ring-shaped smooth muscles and elastic fiber. Tunica
adventitia consists of mainly connective-tissue linking vein with
neighboring tissue.

[0005]Blood endothelial cell layer plays a role in secreting various
physiologically active substances, and controlling blood extension,
blockage of thrombosis, the permeation and transfer of selective
metabolites in vessel wall, and blood flow and adhering white blood cell
and thrombocytic cell with the cell surface. Angioblast, a precursor of
endothelium produced from mesoderm during development stage is
differentiated into various blood endothelial cells in various organs of
human body. Those organ-specific endothelia are presumed to be caused by
surrounding cells or environmental factors in organ (Jain R K., Nat.
Med., 9, pp. 685-693, 2003).

[0006]Various factors occurring in blood endothelial cell layer, for
examples, physical factors such as pulsatile stress on blood cell wall or
the tension on blood vessel; various humoral factors such as excessively
abundant lipid and glucose in blood; and other factors such as the
depletion of oxygen and nutrients caused by blood occlusion or active
oxygen etc, causes to the cell death of blood endothelial cell,
abnormality of tight junction complex and early-aging etc. There have
been reported that the abnormality of blood vessel function results in
the main causes of various human diseases such as vasculitis symptoms,
myocardial infarction, ischemic brain injury, diabetic retinitis etc.
Therefore, the substance preventing from the death and the damage of
endothelial cells such as the depletion of tight-junction and enhancing
the homeostasis of blood cell function may be used usefully in prevention
and treatment of various vascular diseases (O'Riordan E et al., Kidney
Int., 67(5), pp. 1654-1658, 2005; Oda M et al., Clin Hemorheol Microcir.,
23, pp. 199-211, 2000; Brandes R P et al., Cardiovasc. Res., 66(2), pp.
286-294, 2005). Especially, there has been reported that both of
retinopathy of prematurity in infant and diabetic retinopathy in adult,
representative severe diseases in eye occur by the mechanism as follows:
retina blood barrier is destroyed by the action of various factors such
as VEGF released from ischemic premature retina blood and the injury of
angio-endothelial cell in case of retinopathy of prematurity (Gariano R F
et al., Surv. Opthalmol., 40 pp 481-90, 1996); and the permeation of
retinal blood is increase resulting from the destruction of retinal blood
barrier retina and retinal ischemia occurs due to the injury of
angio-endothelial cell in case of diabetic retinopathy (Cogan D G et al.,
Arch. Ophthalmology, 66, pp 366-78, 1961; Palmer E A et al.,
Ophthalmology 98, pp 1620-4-, 1991). Therefore, there have been needed to
develop new agent to inhibit the destruction of retinal blood barrier and
prevent the injury of angio-endothelial cell till now.

[0007]It is known that there are many genus of Panax genus plants belonged
to Araliaceae, for example, Panax ginseng distributed or cultivated in
far-eastern Asia region, Panax quinquefolia in America and Canada, Panax
notoginseng in China, Panax trifolia in eastern region of north America,
Panax japonica in Japan, China and Nepal, Panax pseudoginseng in Nepal,
Panax vietnamensis in Vietnam, Panax elegatior, Panax wangianus and Panax
bipinratifidus etc.

[0008]It has been known that the main constituent of Panax genus plant is
dammarane-skeleton type saponins, such as ginsenosides Rb1,
Rb2, Rc, Rd, Rg1 and Re etc. The biological activities of them are
different from each other in accordance with their chemical structures
and ginseng has been reported to be effective in fatigue recovery or
vigor reinforcement from long years ago.

[0009]Recently, there have been several attempts to strengthen
pharmacological effects of ginseng by modifying the method of ginseng
processing for example, Park et al developed new methods for preparing a
processed ginseng under specific high temperature and high pressure as
disclosed in Korean Patent Registration No. 192678 and U.S. Pat. No.
5,776,460, which changes main ginseng saponins such as ginsenosides Rb1,
Rb2, Rc and Rd, into new saponin metabolites such as ginsenosides Rg3,
Rg5, and Rk1 showing new and more potent pharmacological effects, for
examples, anti-oxidative activity, anti-cancer activity and alleviating
activity of blood circulation etc (Kim W Y et al., J. Nat. Prod., 63(12),
pp. 1702-1704). Especially, the pharmacological effect of ginsenosides
Rg3, Rg5 and Rk1 has known to be strongest among them and those
ginsenosides produced through said new processing method are formed by
the process that a part of sugar moiety in dammarane glycoside, i.e.
ginsenosides Rb1, Rb2, Rc and Rd, was cleaved and continuously subjected
to dehydration reaction at the position of C-20 (See FIG. 1).
Accordingly, these new metabolites can be produced in the root, stem or
leaf of any panax genus plants such as Panax ginseng, Panax quinquefolia,
Panax notoginseng, Panax trifolia, Panax japonica, Panax pseudoginseng,
Panax vietnamensis, Panax elegatior, Panax wangianus and Panax
bipinratifidus which contains dammarane glycoside through the processing
method of Park et al (Korean Patent Registration No. 192678 and U.S. Pat.
No. 5,776,460).

[0010]However, there has been not reported or disclosed about the treating
or preventing effect of the processed ginseng extract prepared by
above-described methods on the disease caused by vascular damage in any
of above cited literatures, the disclosures of which are incorporated
herein by reference.

[0011]Accordingly, the present inventors have discovered that the
processed ginseng product as to make a ratio of ginsenoside (Rg3+Rg5+Rk1)
to (Rb1+Rb2+Rc+Rd) of over 1.0 and the extract thereof show potent
suppressive effect on endothelial dysfunction and the damage of
blood-retinal barrier and finally completed the present invention.

SUMMARY OF THE INVENTION

[0012]The present invention also provides a use of extract of processed
ginseng and saponin compounds isolated therefrom for the prevention or
treatment of the diseases caused by vascular injury.

[0013]The present invention provides a pharmaceutical composition and a
health food comprising extract of processed ginseng and saponin compounds
isolated therefrom as an active ingredient in an effective amount to
treat and prevent the diseases caused by vascular injury.

[0014]The present invention also provides a method for treating or
preventing of the diseases caused by vascular injury in human or mammal
comprising administrating to said mammal an effective amount of above
extract of processed ginseng and saponin compounds isolated therefrom,
together with a pharmaceutically acceptable carrier thereof.

DISCLOSURE OF THE INVENTION

[0015]In accordance with the present invention, the present invention
provides a use of extract of processed ginseng and the saponin compounds
isolated therefrom for the preparation of medicament employed for
preventing or treating the diseases caused by vascular injury in human
and mammals.

[0016]In accordance with the present invention, the present invention also
provides a composition comprising an extract of processed ginseng and
saponin compounds isolated therefrom as an active ingredient in an
effective amount to treat and prevent the diseases caused by vascular
injury.

[0017]The present invention also provides a method for treating or
preventing of the diseases caused by vascular injury in human or mammal
comprising administrating to said mammal an effective amount of an
extract of processed ginseng and saponin compounds isolated therefrom,
together with a pharmaceutically acceptable carrier thereof.

[0018]The term "an extract of processed ginseng" disclosed herein
comprises the extract of Panax genus plants so as to make a ratio of
ginsenoside (Rg3+Rg5+Rk1) to (Rb1+Rb2+Rc+Rd) of over 1.0 as an active
ingredient in an effective amount to treat and prevent the diseases
caused by vascular damage and an extract of processed ginseng obtained by
the steps; treating Panax genus plants with heat at the temperature
ranging from 70 to 150° C. for the period ranging from 2 to 6, as
an active ingredient in an amount effective to treat or prevent the
diseases caused by vascular damage, which can be prepared by the
procedure disclosed in Korean Patent Registration No. 192678 and U.S.
Pat. No. 5,776,460).

[0019]The term ginsenoside "Rg3" disclosed herein includes two isomers of
ginsenoside (20-S) and (20-R).

[0020]The term "saponin compounds isolated from the extract of processed
ginseng" disclosed herein comprise at least one saponin or their
combination selected from the group consisting of ginsenoside Rg3, Rg5
and Rk1.

[0028]For the present invention, for example, dried plant material of
Panax genus, for examples, the root of Panax ginseng is cut into small
pieces and the piece was heated at the temperature ranging from 70 to
150° C., preferably from 100 to 130° C., for the period
ranging from 2 to 6, preferably 3 to 5 hrs; and was mixed with 1 to
20-fold, preferably, 3 to 10-fold weight of water, C1-C4 lower
alcohol such as methanol, ethanol, butanol, or the mixtures thereof,
preferably ethanol; and was heated for the period ranging from 3 to 10
hours, preferably 3 to 6 hours, by reflux extraction with water, cold
water extraction, ultra-sonication or conventional extraction, preferably
by reflux extraction with water; the residue was filtered and then the
filtrate was dried at the temperature ranging from 40 to 80, preferably
from 50 to 70, to obtain inventive extract of processed ginseng.

[0029]To obtain purposed ginsenosides from the inventive extract of
processed ginseng, following procedure could be performed:

[0030]The inventive extract of processed ginseng prepared by above step,
is mixed with water, and then is extracted with 1 to 100-fold,
preferably, 1 to 5-fold volume of non-polar organic solvent such as
hexane, ether, dichloromethane, chloroform, ethyl acetate or the mixtures
thereof; the water-soluble layer obtained the above step is further
extracted with polar organic solvent such as butanol; and the above
organic solvent-soluble extract is further subjected to chromatography
for increasing the content of ginsenoside by repeating the above
extraction steps. The purposed ginsenosides of the present invention,
i.e., ginsenoside Rg3, Rg5 or Rk1 can be obtained by repeated
crystallization steps with appropriate solvent such as water, lower
alcohol, lower ketone, chloroform or the mixture thereof.

[0031]Through the above described procedure, the saponins such as
ginsenoside Rb1, Rb2, Rc, Rd etc being contained in plant
material is transformed into chemically modified ginsenosides such as
ginsenoside Rg3, Rg5, Rk1 etc due to heat treatment.

[0032]In particular, the extract of processed ginseng according to the
present invention wherein a ratio of ginsenoside (Rg3+Rg5+Rk1) to
(Rb1+Rb1+Rc+Rd) of over 1.0 showing more potent physiological activities
than those of extract of non-processed ginseng.

[0033]In order to perform purposed use of the present invention, the use
of the present invention may be embodied by following inventive
composition. The inventive composition may additionally comprise
conventional carrier, adjuvants or diluents in accordance with a using
method. It is preferable that said carrier is used as appropriate
substance according to the usage and application method, but it is not
limited. Appropriate diluents are listed in the written text of
Remington's Pharmaceutical Science (Mack Publishing co, Easton Pa.).

[0034]Hereinafter, the following formulation methods and excipients are
merely exemplary and in no way limit the invention.

[0035]The composition according to the present invention can be provided
as a pharmaceutical composition containing pharmaceutically acceptable
carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose,
sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia
rubber, alginate, gelatin, calcium phosphate, calcium silicate,
cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy
benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral
oil. The formulations may additionally include fillers,
anti-agglutinating agents, lubricating agents, wetting agents, flavoring
agents, emulsifiers, preservatives and the like. The compositions of the
invention may be formulated so as to provide quick, sustained or delayed
release of the active ingredient after their administration to a patient
by employing any of the procedures well known in the art.

[0036]For example, the compositions of the present invention can be
dissolved in oils, propylene glycol or other solvents that are commonly
used to produce an injection. Suitable examples of the carriers include
physiological saline, polyethylene glycol, ethanol, vegetable oils,
isopropyl myristate, etc., but are not limited to them. For topical
administration, the compounds of the present invention can be formulated
in the form of ointments and creams.

[0038]The composition of the present invention in pharmaceutical dosage
forms may be used in the form of their pharmaceutically acceptable salts,
and also may be used alone or in appropriate association, as well as in
combination with other pharmaceutically active compounds.

[0039]The desirable dose of the inventive extract or composition varies
depending on the condition and the weight of the subject, severity, drug
form, route and period of administration, and may be chosen by those
skilled in the art. However, in order to obtain desirable effects, it is
generally recommended to administer at the amount ranging 0.01-10 g/kg,
preferably, 1 to 5 g/kg by weight/day of the inventive extract or
compounds of the present invention. The dose may be administered in
single or divided into several times per day. In terms of composition,
the complex herbal composition should be present between 0.01 to 80% by
weight, preferably 0.5 to 50% by weight based on the total weight of the
composition.

[0040]The pharmaceutical composition of present invention can be
administered to a subject animal such as mammals (rat, mouse, domestic
animals or human) via various routes. All modes of administration are
contemplated, for example, administration can be made orally, rectally or
by intravenous, intramuscular, subcutaneous, intracutaneous, intrathecal,
epidural or intracerebroventricular injection.

[0041]The present inventors demonstrated that the inventive extract of the
present invention showed more potent activity of the disease caused by
vascular injury than that of the extract of processed ginseng prepared by
conventional method or simple processed method, by accomplishing in vitro
and in vivo experiment, e.g., suppressive effect on endothelial
dysfunction and blood-retinal barrier permeation etc., therefore, it has
been confirmed that above described inventive composition is very useful
in the prevention or treatment of the disease caused by vascular injury.

[0042]In particular, the composition of the present invention also potent
preventing activity of the diseases caused by vascular injury, thus it is
very useful for patients susceptible with the various diseases caused by
vascular injury.

[0043]Accordingly, it is another object of the present invention to
provide a health care food comprising above described extract or saponin
compounds isolated therefrom prepared by above processes and a
sitologically acceptable additive to prevent or alleviate the diseases
caused by vascular injury.

[0044]The present invention also provides a health care food comprising
the extract of processed ginseng obtained by the steps; heating Panax
plant with heat at the temperature ranging from 70 to 150° C. for
the period ranging from 2 to 6, and a sitologically acceptable additive
to prevent or alleviate the diseases caused by vascular injury.

[0045]The present invention also provides a health care food comprising
saponin compounds at least one or their combination selected from the
group consisting of ginsenoside Rb3, Rg5 and Rk1 and a sitologically
acceptable additive to prevent or alleviate the diseases caused by
vascular injury.

[0046]Above-described composition therein can be added to food, additive
or beverage for prevention of the diseases caused by vascular injury. For
the purpose of preventing the diseases caused by vascular injury,
wherein, the amount of above described extract or compound in food or
beverage may generally range from about 0.1 to 15 w/w %, preferably 1 to
10 w/w % of total weight of food for the health food composition and 1 to
30 g, preferably 3 to 10 g on the ratio of 100 m of the health beverage
composition.

[0047]Providing that the health beverage composition of present invention
contains above described extract or compounds as an essential component
in the indicated ratio, there is no particular limitation on the other
liquid component, wherein the other component can be various deodorant or
natural carbohydrate etc such as conventional beverage. Examples of
aforementioned natural carbohydrate are monosaccharide such as glucose,
fructose etc; disaccharide such as maltose, sucrose etc; conventional
sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol,
and erythritol etc. As the other deodorant than aforementioned ones,
natural deodorant such as taumatin, stevia extract such as levaudioside
A, glycyrrhizin et al., and synthetic deodorant such as saccharin,
aspartam et al., may be useful favorably. The amount of above described
natural carbohydrate is generally ranges from about 1 to 20 g, preferably
5 to 12 g in the ratio of 100 m of present beverage composition.

[0048]The other components than aforementioned composition are various
nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring
agent, a coloring agent and improving agent in case of cheese chocolate
et al., pectic acid and the salt thereof, alginic acid and the salt
thereof, organic acid, protective colloidal adhesive, pH controlling
agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent
used in carbonate beverage et al. The other component than aforementioned
ones may be fruit juice for preparing natural fruit juice, fruit juice
beverage and vegetable beverage, wherein the component can be used
independently or in combination. The ratio of the components is not so
important but is generally range from about 0 to 20 w/w % per 100 w/w %
present composition.

[0050]Inventive extract or compound of the present invention has no
toxicity and adverse effect therefore they can be used with safe.

[0051]The present invention is more specifically explained by the
following examples. However, it should be understood that the present
invention is not limited to these examples in any manner.

BRIEF DESCRIPTION OF DRAWINGS

[0052]The above and other objects, features and other advantages of the
present invention will more clearly understood from the following
detailed description taken in conjunction with the accompanying drawings,
in which;

[0053]FIG. 1 shows the structural change of ginsenoside due to heat
treatment of Panax plant,

[0055]FIG. 3 is the result of MTT assay showing suppressive effect of SG
on the apoptosis of HUVEC (a); of SG (b) and of ginsenosides Rb1, Rg1,
Re, Rg3, Rg5, Rk1 on retinal vascular endothelial cell (c),

[0056]FIG. 4 is the result of MTT assay showing suppressive effect of Rk1
on the apoptosis of HUVEC in medium,

[0057]FIG. 5 is the result of staining with DAPI showing suppressive
effect of Rk1 on the apoptosis of HUVEC in medium,

[0058]FIG. 6 is the result of western blot analysis showing recovery
effect of Rk1 on the decrease of tight junction protein induced by
vascular endothelial growth factor and by AGE on human retinal
endothelial cell (a) and recovery effect of Rk1 on the tight junction
protein of mouse retina (b),

[0059]FIG. 7 is the result of immuno-cytochemical staining method showing
recovery effect of Rk1 on the decrease of tight junction protein caused
by AGE on human retina endothelial cell,

[0060]FIG. 8 is the result of immuno-cytochemical staining method showing
recovery effect of Rk1 on the decrease of tight junction protein caused
by vascular endothelial growth factor on human retinal endothelial cell,

[0061]FIG. 9 is the result of immuno-cytochemical staining method showing
recovery effect of Rk1 on the decrease of tight junction protein caused
by vascular endothelial growth factor on mouse retina,

[0062]FIG. 10 is the result of immuno-cytochemical staining method showing
recovery effect of Rk1 on the decrease of tight junction protein caused
by diabetes on mouse retina,

[0063]FIG. 11 is the result of angiography analysis showing recovery
effect of Rk1 on the permeability increase of blood vessel in retina
caused by vascular endothelial growth factor and diabetes on mouse
retina.

BEST MODE FOR CARRYING OUT THE INVENTION

[0064]It will be apparent to those skilled in the art that various
modifications and variations can be made in the compositions, use and
preparations of the present invention without departing from the spirit
or scope of the invention.

[0065]The present invention is more specifically explained by the
following examples. However, it should be understood that the present
invention is not limited to these examples in any manner.

EXAMPLES

[0066]The following Reference Example, Examples and Experimental Examples
are intended to further illustrate the present invention without limiting
its scope.

[0068]1 Kg of dried plant material of Panax genus, for examples, the root
of Panax ginseng was cut into small pieces and the sliced piece was
heated at 120° C. for 4 hours in autoclave. The processed ginseng
was mixed with 2 of ethanol and heated for 4 hours by reflux extraction
with water. The residue was filtered and then the filtrate was evaporated
to obtain inventive extract of processed ginseng, which designated as
`SG.` hereinafter.

Example 2

Preparation of Ginsenoside Rg3, Rg5 and Rk1 Isolated from the Extract of
Processed Ginseng

[0069]10 g of processed ginseng extract prepared by the above Example 1
was mixed with 100 m of water, and then was extracted with 100 m of ether
3 times. The remaining water-soluble layer was further extracted with 100
m of butanol 3 times. The butanol soluble extract was further subjected
to silica gel column chromatography and eluted with a
ethylacetate:methanol:water mixture (20:1:1) to obtain 500 mg of the
fraction containing ginsenoside Rg3 by repeating the above chromatography
procedure. The fraction containing ginsenosides Rg5 and Rk1 was further
purified over semi-preparative HPLC using reverse phase column with 60%
CH3CN eluent. 200 mg of Rg5 and 150 mg of Rk1 were obtained.

[0072]HUVEC and retinal vascular endothelial cell were spread on 24-well
plates (3×104 cells/well) and were incubated for 24 hours. The
mediums were substituted with new medium containing 20% (w/v) FBS and FBS
free medium respectively. The FBS free medium was treated with various
concentrations of SG, i.e., 0, 1, 5, 10, 20 μg/m. The medium was
incubated for 24 hours. After the incubation, MTT assay was performed.

[0073]As shown in FIG. 3a and FIG. 3b, SG inhibited the apoptosis of HUVEC
and retinal vascular endothelial cells and increased the cell viability
of HUVEC and retinal vascular endothelial cells in a dose dependent
manner.

[0075]HUVEC and retinal vascular endothelial cell were spread on 24-well
plates (3×104 cells/well) and were incubated for 24 hours
respectively. The mediums were substituted with new medium containing 20%
(w/v) FBS and FBS free medium respectively. The FBS free medium was
treated with 10 μg/m of SG, Rb1, Rg1, Re, Rg3, Rg5 and Rk1 and the
medium was incubated for 24 hours. After the incubation, MTT assay was
performed.

[0076]As shown in FIG. 3c, SG, Rg3, Rg5 and Rk1 inhibited the apoptosis of
HUVEC and retinal vascular endothelial cells and increased the cell
viability of HUVEC and retinal vascular endothelial cells in a dose
dependent manner while Rb1, Rg1 and Re did not.

1-4. Suppressive Effect of Rk1 on the Cell Apoptosis of Vascular
Endothelial Cell Caused by the Lack of Serum

[0077]Vascular endothelial cell were spread on 24-well plates
(3×104 cells/well) and were incubated for 24 hours. The
mediums were substituted with new medium containing 20% (w/v) FBS and FBS
free medium respectively. The FBS free medium was treated with various
concentrations of Rk1, i.e., 0, 0.5, 1, 5, 10 μg/m and incubated for
24 hours. After the incubation, MTT assay was performed.

[0082]The cells were transferred to human endothelial SFM-basal growth
medium (Invitrogen, USA), incubated for 24 hours and transferred to new
human endothelial SFM-basal growth medium again. The medium was treated
with 20 ng/m of VEGF (vascular endothelial growth factor) and Rk1, and
incubated for 6 hours. After the incubation, the cells were collected to
induce lysis and western blot analysis was performed.

[0083]As shown in FIG. 6a, Rk1 show potent recovering effect on the
decrease of all tight junction proteins, i.e., ZO-1, ZO-2 and occludin
which was caused by VEGF and AGE (advanced glycation end product).

2-2. Recovery Effect of Rk1 on the Decrease of Tight Junction Protein
Caused by Vascular Endothelial Growth Factor and Diabetes

[0084]Both of 100 ng of VEGF and 30 μg of Rk1 were injected into the
retina of C57/BL6 mice (Orient, Korea). After 24 hours, the extracted
retina from mice was induced to lysis and western blot analysis was
performed.

[0085]As shown in FIG. 6b, Rk1 showed potent recovering effect on the
decrease of all tight junction proteins, i.e., ZO-1, ZO-2 and occludin
which was caused by VEGF and diabetes.

2-3. Immunocytochemistry Analysis (1)

[0086]Human retinal vascular endothelial cell were spread on 35 mm dish
(2×105 cells/well) and were incubated for 24 hours. The cells
were transferred to human endothelial SFM-basal growth medium
(Invitrogen, USA), incubated for 24 hours and transferred to new human
endothelial SFM-basal growth medium again. The medium was treated with 20
ng/m of VEGF (vascular endothelial growth factor) and Rk1, and incubated
for 6 hours. After the incubation, the medium was washed with PBS
solution and immunocytochemistry analysis was performed to determine the
change of tight junction protein.

[0087]As shown in FIG. 7 and FIG. 8, Rk1 showed potent recovering effect
on the decrease of tight junction protein, i.e., ZO-1, ZO-2 and occludin
which was caused by VEGF and AGE (advanced glycation end product).

2-4. Immunocytochemistry (2)

[0088]Both of 100 ng of VEGF and 30 μg of Rk1 were injected into the
retina of C57/BL6 mice (Orient, Korea). After 24 hours, retina was
delivered from the mice and western blot analysis was performed.

[0089]As shown in FIG. 9 and FIG. 10, Rk1 showed potent recovering effect
on the decrease of tight junction protein, i.e., ZO-1, ZO-2 and occludin
which was caused by VEGF and diabetes.

2-5. Angiography

[0090]Both of 100 ng of VEGF and 30 μg of Rk1 were injected into the
retina of C57/BL6 mice (Orient, Korea). After 24 hours, the mice were
anesthetized and FITC-dextran was injected into the heart to allow
fluorescent material to flow in blood. After 30 minutes, the extracted
retina from mice was performed to flat mounting and the change was
observed with fluorescent microscope. In the only VEGF-injected mice, it
has been confirmed that the fluorescent material was exuded from
neighboring tissues around blood vessel caused by the increased
permeability of retinal vessel however the exudation in VEGF was
recovered by the treatment of Rk1.

[0091]Likewise, it has been also confirmed that the fluorescent material
was exuded from neighboring tissues around blood vessel caused by the
increased permeability of retinal vessel in diabetes induced mice however
the exudation was recovered by the treatment of Rk1.

[0092]Hereinafter, the formulating methods and kinds of excipients will be
described, but the present invention is not limited to them. The
representative preparation examples were described as follows.

Injection preparation was prepared by dissolving active component,
controlling pH to about 7.5 and then filling all the components in 2 m
ample and sterilizing by conventional injection preparation method.

[0100]Health beverage preparation was prepared by dissolving active
component, mixing, stirred at 85° C. for 1 hour, filtered and then
filling all the components in 1000 m ample and sterilizing by
conventional health beverage preparation method.

[0101]The invention being thus described, it will be obvious that the same
may be varied in many ways. Such variations are not to be regarded as a
departure from the spirit and scope of the present invention, and all
such modifications as would be obvious to one skilled in the art are
intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

[0102]As described in the present invention, the extract of processed
Panax genus plant and saponin compounds isolated therefrom show
suppressive effect on the endothelial dysfunction and the injury of
blood-retinal on HUVEC and retinal vascular endothelial cell. Therefore,
it can be used as the therapeutics or functional health food for treating
and preventing the diseases caused by vascular injury.